Survival Strategies of the Rotifer Brachionus rotundiformis - E-FAS

Original Article
Fish Aquat Sci 15(1), 57-62, 2012
Survival Strategies of the Rotifer Brachionus rotundiformis
for Coexisting with the Copepod Apocyclops borneoensis in
Laboratory Culture
Min-min Jung*
Future Aquaculture Research Center, National Fisheries Research and Development Institute, Jeju 690-192, Korea
Abstract
Interspecific relationship between a euryhaline rotifer Brachionus rotundiformis and a cyclopoid copepod Apocyclops borneoensis was investigated in the laboratory culture. In a mixed culture of B. rotundiformis and A. borneoensis, population growth of
B. rotundiformis was suppressed from day 10, while growth in a monoculture population continuously increased throughout the
experimental period. However, the population growth of A. borneoensis in the mixed culture did not markedly differ from that
in a monoculture population. Suppression of B. rotundiformis growth coincided with a decrease in the numbers of both non-eggbearing and egg-bearing females, and increasing resting egg formation. Growth of A. borneoensis was not affected by the presence
of the rotifer. However, relative growth index of ovisac bearing females in the mixed culture was 1.62 times higher than that in
the monoculture. Presence of the copepod did not greatly reduce the food available to the rotifer population. The rotifer B. rotundiformis responded in a unique way, to stresses such as physical damage (filtering by A. borneoensis) with the production of many
resting eggs to increase its chances of survival.
Key words: Apocyclops borneoensis, Brachionus rotundiformis, Cyst, Interspecific relation, Resting egg, Rotifer Introduction
Rotifers are some of the most important zooplankton, and
are the focus of much attention from aquaculture scientists.
Rotifers have been used as a primary live food for the seedling
production of many economically important marine animals,
because rotifers can be easily cultured at high densities. Stable
mass culture of rotifers is needed for successful fish larval
rearing. A variety of mass culture methods should be developed to facilitate successful aquatic animal culture through
stable live food organism production. However, the instability
or sudden crashes of rotifer mass cultures remain problematic.
The causes of such culture failures are not fully understood,
although it is predicted that one major cause is contamination by other organisms such as protozoa (Takayama, 1979;
Reguera, 1984; Chen et al., 1997), copepods (Fukusho et al.,
Open Access
1976) and bacteria (Yu et al., 1990).
Coexing populations of different taxonomic groups depend
on the organisms that occur together in space and time, and
interact with each other through the processes of mutualism,
parasitism, predation and competition (Begon et al., 1990).
The relationships between Brachionus rotundiformis and
other zooplankton species have been well examined (Gilbert
and Stemberger, 1985; Gilbert, 1988; Hagiwara et al., 1995a;
Jung et al., 1997), and competitive interference has been reported between rotifers and cladocera in fresh water. Under
marine culture conditions, there is considerable evidence for
interspecific relationships between a rotifer, B. rotundiformis,
and two species of copepods, Tigriopus japonicus and Acartia
sp. (Jung et al., 1997).
Received 21 July 2011; Revised 6 September 2011;
Accepted 9 February 2012
http://dx.doi.org/10.5657/FAS.2012.0057
This is an Open Access article distributed under the terms of the Creative
Commons Attribution Non-Commercial License (http://creativecommons.
org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use,
distribution, and reproduction in any medium, provided the original work
is properly cited.
pISSN: 2234-1749 eISSN: 2234-1757
Copyright © The Korean Society of Fisheries and Aquatic Science
*Corresponding Author
E-mail: jminmin@nfrdi.go.kr
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Fish Aquat Sci 15(1), 57-62, 2012
A
B
C
Fig. 1. Experimental two organisms rotifer, Brachionus rotundiformis (A) and copepod, Apocyclops borneoensis (B) and interspecific relationship between B.
rotundiformis and A. borneoensis (C).
rotundiformis as a Bitung strain.
Experimental design and conditions used were the same as
those described by Hagiwara et al. (1995b). Salinity, temperature, and culture volume were 22 psu, 25ºC and 40 mL, respectively. The organisms were cultured in total darkness. The
initial number of animals in mixed cultures was 20 females
of the Bitung rotifer strain of B. rotundiformis and 3 ovisacbearing females of A. borneoensis. In the monocultures, the
numbers of rotifers and copepods were the same as in mixed
cultures, but were cultured separately. Mono- and mixed-species cultures were conducted with three replicates for 16 days.
Stereo-microscopic observation was carried out on fresh culture medium including Tetraselmis suecica (7 × 105 cells/mL)
every two days. Total number of test animals was counted and
remaining algal food density was also counted by a haemacytometer (Kayagaki Irika Kogyo Co. Ltd., Tokyo, Japan). The
algal food T. suecica, was grown in modified Erd-Schreiber
medium (Hagiwara et al., 1994) and centrifuged. Density of
food added was 7 × 105 cells/mL, and was readjusted every
two days after observation.
For the observation and calculation of the rotifer mixis
rate (%), all individual non-egg bearing females, amictic females, unfertilized and fertilized mictic females, males and
resting eggs were counted, and the mixis rate was calculated
(Hagiwara et al., 1988). The numbers of all individual nauplii,
copepodites and egg-bearing females of the copepod A. borneoensis were recorded. Relative population growths between
monocultures of each species and mixed culture conditions
were compared by student's t-test.
Copepods and rotifers coexist in estuaries as well as in
brackish water fish culture ponds in North Sulawesi, Indonesia. Of several copepod species collected, the only one that
could be adapted to laboratory culture was Apocyclops borneoensis. It is unknown which species (B. rotundiformis or A.
borneoensis) dominates earlier, or which is more susceptible
to the rigorous conditions of the ponds. Therefore, it is of interest to examine the nature of the relationship between these
two species. Such information is useful both for assessing
their ecological significance in a brackish water ecosystem, as
well as for establishing techniques for mono- and mixed-species cultures for aquaculture purposes as live food organisms.
Here, I observed, the survival strategies involved in the interspecific relationship between B. rotundiformis and A. borneoensis under laboratory experimental conditions. I focused
on the interspecific interactions of the rotifer and the copepod
from a microcosm viewpoint of aquaculture ecology.
Materials and Methods
Copepods and rotifers were collected from a milkfish pond
in Bitung, 30 km east of Manado, North Sulawesi, Indonesia.
The pond is separated from the adjacent sea by mangroves,
but is connected through an inlet during high tide. Throughout
the year, salinity of the pond varies from 12 to 25 psu and
temperature ranges from 29ºC to 35ºC.
The specimens were kept in darkness during a three-day
acclimation culture to laboratory conditions before isolation.
Various copepods were included in the sample, but only a
cyclopoid copepod survived. The species was identified as
Apocyclops borneoensis by Dr. H-S. Kim (Research Institute
for Basic Science, Cheju National University of Korea). The
rotifers were morphologically analyzed by Fu et al. (1991),
and evidently belonged to an ultra minute strain of Brachionus
rotundiformis (Hagiwara et al., 1995a). We refer this rotifer B.
http://dx.doi.org/10.5657/FAS.2012.0057
Results
Predator-prey interactions were not observed during this
experiment between the experimental rotifer B. rotundiformis and copepod A. borneoensis (Fig. 1A and 1B). However,
58
Jung (2012) Cyst Formation of Rotifer for Survival of Them
10
1,600
A
1,200
8
No. of rotifers
Remained microalgae
(10 5cells/mL)
12
6
4
2
0
800
400
0
2
4
6
8
10
12
14
16
Days
Remained microalgae
(10 5cells/mL)
12
10
0
2
4
6
8
10
12
14
16
Days
B
Fig. 3. Population growth of rotifer Brachionus rotundiformis (Bitung
strain) in the mono culture (white circles) and mixed culture with copepod
Apocyclops borneoensis (black circles).
8
6
4
80
2
0
70
0
2
4
6
8
10
12
14
60
16
12
10
Mixis rate (%)
Days
Remained microalgae
(105cells/mL)
0
C
40
30
20
8
10
6
0
4
0
2
4
6
8
Days
10
12
14
16
Fig. 4. Comparison of mixis rates in the rotifer Brachionus rotundiformis
2
0
50
0
2
4
6
8
10
12
14
(Bitung strain) mono culture (white circles) and mixed culture with
copepod Apocyclops borneoensis (black circles).
16
Days
Fig. 2. Densities of remaining food on the every two days in rotifer
Brachionus rotundiformis mono culture (A), copepod Apocyclops
borneoensis mono culture (B) and two species (rotifer and copepod)
mixed culture (C).
Table 1. Comparison of production of rotifer Brachionus rotundiformis
resting eggs in the rotifer mono culture and mixed culture with copepod
Apocyclops borneoensis
distinct correlation interactions between the two experimental
organisms were observed (Fig. 1C).
The remnant food (T. suecica) volumes under rotifer culture
conditions significantly decreased with the lapse of experimental time compared to those in copepod monoculture condition (P < 0.05) (Fig. 2A and 2C), and almost all microalgae
(T. suecica) cells were consumed. A remnant food volume of
approximately 7 × 105 cells/mL was maintained under copepod monoculture conditions (Fig. 2B).
Population growth of rotifers in the mixed culture was significantly decreased (P < 0.05) (Fig. 3). Growth of rotifers
was suppressed by the presence of copepods in the culture
container. However, the population growth of rotifers in the
monospecific culture continually increased (Fig. 3).
In both experimental conditions, inductions of mixis rates
(%) were observed. However, the mixis rate (%) of rotifers
was notably changed by presence or absence of copepods.
The difference between rotifer monoculture and mixed culture
No. of resting eggs
Tank No. 1
Tank No. 2
Tank No. 3
Mean ± SD
Mono
704
764
1,093
853 ± 171
Mixed
1,568
1,050
1,365
1,327 ± 213
with copepods was particularly notable on the 4th day (Fig. 4).
The presence of copepods influenced the mixis rate (%) of coexisting rotifers (Fig. 4), as well as the production numbers of
rotifer resting eggs. On day 4 of the observations, the highest
values of mixis rates (%) was observed in both experimental
conditions (Fig. 4), and the production of rotifer resting eggs
was observed from the 6th day. However, there were differences in the numbers of rotifer resting eggs produced. More
resting eggs were formed in the copepod mixed culture than
in the rotifer monoculture (P < 0.05) (Fig. 5). A comparison
of the production of rotifer resting eggs between rotifer monoculture and mixed culture with copepods is shown in Table 1.
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Fish Aquat Sci 15(1), 57-62, 2012
500
120
400
100
Nauplii
No. of rotifer resting eggs
140
80
60
40
0
0
2
4
6
8
10
12
14
2
4
6
8
10
12
14
16
10
12
14
16
10
12
14
16
Days
300
Fig. 5. Formed numbers of rotifer resting eggs in the rotifer Brachionus
250
Copepodites
rotundiformis (Bitung strain) mono culture (white circles) and mixed
culture with copepod Apocyclops borneoensis (black circles).
800
700
200
150
100
50
600
0
500
0
2
4
6
8
Days
400
80
300
Egg-carrryng females
Number of copepods
0
16
Days
200
100
0
200
100
20
0
300
0
2
4
6
8
10
12
14
16
Days
Fig. 6.
Population growth of copepod Apocyclops borneoensis in the
mono culture (white circles) and mixed culture with rotifer Brachionus
rotundiformis (Bitung strain, black circles).
70
60
50
40
30
20
10
0
0
2
4
6
8
Days
Fig. 7. Comparison of the numbers of each developmental stages,
The production of rotifer resting eggs was 1.56 times higher
in the mixed culture than in the rotifer monoculture (P < 0.05)
(Table 1), with 1,327 ± 213 (mean ± SD) resting eggs in mixed
cultures, and 853 ± 171 (mean ± SD) in monocultures. Fig. 6 shows that copepod population growth did not differ
between copepod monoculture and mixed culture with rotifers during the 16 day experimental period. This trend was
not changed with the separation of the developmental stages
(nauplius, copepodid and egg-carrying females) of the copepods (Fig. 7).
nauplius, copepodid and egg carrying female of copepod Apocyclops
borneoensis in the mono culture (blank circles) and mixed culture with
rotifer Brachionus rotundiformis (solid circles).
No predator-prey relationship was observed between the
two zooplankton species during the experimental period.
However, interference between the two species was observed.
It was noted that the copepod used a filter feeding mechanism
in which it filtered the slowly swimming rotifers together with
algae (food), but immediately rejected the rotifers, which
started to swim. Rejected rotifers appeared to be unharmed,
but may have suffered some physical damage or injury. The
physical damage by copepods may have brought about the decrease in rotifer population growth.
Microalgae, including Nannochloropsis, Dunaliella, Tetraselmis and enriched freshwater Chlorella are commonly used
foods in the mass culture of rotifers (Witt et al., 1981). Of
these, Tetraselmis is well known among aquaculturists as a
good food for rotifer and copepod cultures. In this study, in
both mono and mixed cultures of rotifers, the density of T.
suecica continuously decreased. However, in the monoculture
of the copepod A. borneoensis, algal density did not decrease,
Discussion
Rotifer mass culture tanks comprise intricate relationships
among the constituents of the small ecosystem. These small
ecosystems are mainly composed of rotifers and contaminant
micro-organisms, such as bacteria, microalgae, protozoa and
copepods. Many of these contaminant micro-organisms affect
rotifer population growth through interactions such as exploitative competition for food, conmensalism, ammensalism and
physical interference competition (Hagiwara et al., 1995b;
Jung et al., 1997).
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Jung (2012) Cyst Formation of Rotifer for Survival of Them
but maintained a constant level after small amounts were used
as food by the copepods, and the added food volume appeared
sufficient to maintain algal density. This indicated that A. borneoensis may not eat much and/or did not actively feed on
T. suecica in monoculture. This suggests that there was not
competition for food, and that sufficient amounts of food (T.
suecica) were supplied for the copepod A. borneoensis under
these experimental conditions.
The presence of the rotifer did not influence the population growth of the copepods, likely because of the large size
differential between B. rotundiformis (c. 02 mm) and A. borneoensis (c. 0.8 mm). However, the copepod suppressed rotifer population growth after 10 days in mixed culture. The
population growth of B. rotundiformis in mixed culture was
associated with a reduction in the numbers of both non-egg
bearing females and egg-bearing females.
An analysis of the population structures revealed that the
rotifer population growth decreased due to a 30% reduction in
females without eggs and amictic females. In contrast, mictic
females in mixed cultures were more abundant than those in
monocultures. Thus, the presence of copepods stimulated the
sexual reproduction of the rotifer (mixis rate or resting egg
formation), which in turn caused decreased rotifer population
growth. It is of interest to conduct further research to clarify
this mechanism.
The dormant stage of the rotifer (resting egg or cyst) is very
resistant to harsh environmental conditions and may be dispersed over wide areas by wind, water or migrating animals.
Sometimes, scientists induce the production of mixis reproduction (cyst) as an easy method of storing and transporting for marine fish larvae culture or aquaculture study. The
hatching of rotifer resting eggs is caused by stimulation from
light, temperature, salinity and some chemicals (reviewed by
Hagiwara, 1996), but there is no known method to artificially
produce rotifer cysts. The high mixis rate (70% or more) in this study could be
related to the algal species used as food. Tetraselmis tetrathele
as a food source stimulated a high mixis rate (81%), which led
to the successful mass production of resting eggs (101 x 106
Ind.) of the Hawaiian strain of B. rotundiformis (Hagiwara and
Lee, 1991). Another report indicated that bacteria populations
in mass cultures regulated the sexual reproduction of rotifers
(Hagiwara et al., 1994). Two marine copepod species were
successfully cultured when consuming bacteria alone (Rieper,
1978). This mechanism has also been discussed with the effects of bacteria on interspecific relationships between B. rotundiformis and the two copepod species, Tigriopus japonicus
and Acartia (Hagiwara et al., 1995b; Jung et al., 1997). The
present study also highlights the significance of bacterial flora
on the population growth of A. borneoensis.
Population growth of A. borneoensis in mixed culture did
not differ compared with that in monoculture. However, the
number of nauplii decreased to 17% and the number of egg
bearing females was 1.62 times higher in mixed culture. The
reason for this was not clear, but may be associated with the
feeding mechanisms of copepods such as Apocyclops, which
may utilize feces more efficiently than they do bacteria in the
water column. Various copepod species used as live food organisms utilized bacteria as their food. The common marine
copepod species Tigriopus japonicus utilized bacteria (Jung
et al., 1998) and rotifer feces (Jung et al., 2000) as food in the
early nauplius developmental stages. The individuals in each
developmental stage may also have different feeding habitats,
due to changes in their mandible structures (Itoh, 1970). The
experimental copepod Apocyclops appears to utilize egg sac
conservation for the preservation of the species. This may
explain the low survival rate of Apocyclops nauplii that was
observed in mixed cultures.
Acknowledgments
I am indebted to Dr. Cheng-Sheng Lee of Center for Tropical and Subtropical Aquaculture, The Oceanic Institute in Hawaii, USA and Mr. Diegane Ndong of Agence Nationale De
L’Aquaculture, Senegal. This study was funded by National
Fisheries Research & Development Institute of Korea (NFRDI, RP-2011-AQ-059).
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